A novel role of hydrogen peroxide in Kaposi sarcoma-associated herpesvirus reactivation

Abstract

Reactivation of Kaposi sarcoma-associated herpesvirus (KSHV) from latency for lytic replication plays a pivotal role in the development of KS tumors. However, the physiological factors of KSHV reactivation in KS patients remain undefined. Two recent studies independently discovered that the reactive oxygen species (ROS) H2O2 induces KSHV reactivation in latently infected cells, which can be inhibited by H2O2-specific antioxidants. H2O2 not only directly induces KSHV reactivation but also is involved in spontaneous lytic replication as well as reactivation stimulated by TPA, hypoxia, and cytokines. Furthermore, in a xenograft-based primary effusion lymphoma (PEL) mouse model, in vivo KSHV reactivation is also H2O2-dependent and can be suppressed by antioxidants. Mechanistically, H2O2 primarily activates the MAPK pathways to induce viral lytic gene expression and replication. This new finding defines a novel role of H2O2 in KS tumorigenesis and highlights great potentials of using antioxidants and anti-inflammatory drugs for the prevention and treatment of KS tumors.

Hydrogen peroxide (H₂O₂) is a reactive oxygen species (ROS) produced during oxidative stress. It is also produced by inflammatory phagocytes through induction of NADPH oxidases. At high concentration, H₂O₂ causes apoptosis and senescence in primary cells. At lower levels, H₂O₂ activates multiple cellular REDOX signaling pathways including the mitogen-activated protein kinases (MAPK) pathways. The latter are required and sufficient for reactivation of Kaposi sarcoma-associated herpesvirus (KSHV) for lytic replication from latency,1 a viral event that plays a pivotal role in the development of Kaposi sarcoma (KS).2

KS, a vascular malignancy of endothelial cell origin, is etiologically associated with infection by KSHV.3 Like other herpesviruses, the life cycle of KSHV consists of a lytic phase and a latent phase. In healthy people, following an acute infection, KSHV establishes a life-long persistent latent infection in the lymphoid tissues. In immune-compromised patients, KSHV reactivates to undergo lytic replication to produce virions that spread and cause de novo infection, promoting proliferation, angiogenesis and inflammation to result in hyperplasia. If untreated, the hyperplasia may progress to become truly malignant tumors. Thus, KSHV reactivation from the viral reservoir plays a pivotal role in the initiation of KS. Indeed, progression of KS is tightly correlated with viral load.4 In KS tumors, most cells are latently infected by KSHV. A small subset of KS cells undergoes spontaneous lytic replication. These lytic-replicating cells also play important roles in tumor growth, as drugs such as cidofovir (CDV) and ganciclovir (GCV) that specifically inhibit viral replication cause tumor regression and the regressed tumors quickly return upon withdrawal of these drugs.5 Nevertheless, the physiological factors that trigger KSHV reactivation in KS patients remain obscure. To address this question, two recent studies from Dr. Ren Sun's laboratory and our group independently discovered that the ROS H₂O₂ plays a pivotal role in this viral event.6,7

We observed a dose-dependent induction of KSHV lytic gene expression and virion production from latently infected human primary effusion lymphoma (PEL) cell lines and primary human umbilical vein endothelial cells (HUVEC) with exogenous H₂O₂. Higher level of intracellular H₂O₂ resulting from activity inhibition or expressional “knock-down” of catalase, an enzyme that removes intracellular H₂O₂, also elicited a similar effect. Ren Sun's group further demonstrated that inhibition of NFκB pathway increased intracellular H₂O₂ to induce KSHV lytic gene expression and cell death. Consistent with previous finding that NFκB pathway promotes KSHV latency by suppressing lytic replication,8 this result suggested a role of this cellular survival pathway in REDOX balancing as well. Together, results from these two studies confirmed that both exogenous and endogenous H₂O₂ induces KSHV reactivation.

Previously, 12-O-tetradecanoyl phorbol-13-acetate (TPA), hypoxia and inflammatory cytokines have been shown to induce KSHV lytic replication.9–11 We found that these stimuli also increased intracellular H₂O₂ and that antioxidants such as catalase, reduced glutathione and N-acetyle-cysteine (NAC) attenuated viral reactivation induced by these factors. The antioxidants also abolished spontaneous KSHV lytic replication under normal culture conditions. Thus, H₂O₂ not only directly induces KSHV reactivation but also mediates spontaneous lytic replication and reactivation induced by TPA, hypoxia and cytokines. To measure KSHV reactivation in vivo, we generated a recombinant KSHV by replacing the small capsid protein ORF65 with the firefly luciferase gene. We injected BCBL1 cells stably containing this recombinant virus into NOD/SCID mice, and treated the mice with or without 5 mM NAC in drinking water. All mice grew ascites 5 to 6 weeks post-inoculation. NAC-treated mice had significantly lower luciferase activity, lower levels of KSHV lytic proten ORF65 in the ascites fluids and viral loads in the blood than untreated mice. Therefore, KSHV lytic replication in vivo is also H₂O₂-dependent.

Mechanistically, H₂O₂ increased phosphorylation of ERK1, JNK and p38, as well as c-Jun, a subunit of the AP1 complex and a downstream target of the MAPK pathways. Inhibition of each of these MAPK pathways by specific inhibitors or dominant negative constructs blocks H₂O₂ induction of viral gene expression and virion production. Thus, H₂O₂ induces KSHV reactivation through activation of the MAPK and AP1 pathways. Nevertheless, H₂O₂ may also engage other signaling pathways and transcription factors such as HIF-1/2 and XBP-1 for the regulation of KSHV reactivation. Indeed, we found that H₂O₂ induced hypoxiainducible factor-1α (HIF-1α), which is known to upregulate expression of several key KSHV lytic genes through direct binding to the viral promoters.10,11

In summary, due to oxidative stress and chronic inflammation, the higher level of H₂O₂ in KS patients likely plays a pivotal role in KS tumorigenesis through induction of KSHV reactivation, cell proliferation, angiogenesis and inflammation. This new finding not only defines a novel role of H₂O₂ in KSHV pathogenesis but also highlights the great potentials of using antioxidants and anti-inflammatory drugs for the prevention and treatment of KS.

Acknowledgments

The authors declare no conflict of interest. This study was supported by a Pilot Grants Program from the Nathan Shock Center of Excellence in the Biology of Aging from the Barshop Institute for Longevity and Aging Studies at San Antonio, TX to Ye FC and grants from National Institute of Health (DE017333, CA096512, CA124332 and CA119889) to S.J. Gao. We thank Bedolla, Roble G. for proofreading this manuscript and her suggestions.